More than 28 million Americans suffer from various forms of hearing loss and 30 million more are exposed to dangerous levels of noise. The lack of effective treatment for many forms of acquired and inherited hearing disorders has prompted interest in the potential application of newly developed gene delivery techniques to restore normal cochlear function. Towards this goal, a number of vectors have been successfully used to mediate intracochlear gene delivery including liposomes, herpes virus, lentivirus, adenovirus, and adeno-associated virus (AAV). Unfortunately, viral vectors (with the exception of AAV) introduce viral genes into the transduced cells, targeting them for destruction by the immune system and limiting the duration of transgene expression. In addition, many of these vectors have been associated with pathogenic conditions. In contrast, AAV is particularly attractive as a gene delivery system because 96% of its viral genome has been removed. Furthermore, it has never been associated with disease, has a broad host range, and facilitates long term gene expression. Importantly, 6 different AAV serotypes have been isolated (AAV 1-6) which differ in their respective capsid proteins. Consequently these serotypes exhibit moderate to substantial differences in the efficiency of transduction as well as the cell types transduced. AAV-2 has been used in cochlear gene delivery studies where it does not appear to transduce cochlear hair cells. However, AAV-2 requires heparan sulfate on the cell surface for entry into target cells and significantly, hair cells lack heparan sulfate. Therefore, it is not surprising that AAV-2 has not been a successful vector for gene transfer within the cochlea. We propose to systematically evaluate transgene delivery to the mouse cochlea and spiral ganglia using AAV serotypes 1-5 both in vitro and in vivo. Our goal is to determine those cell populations that each AAV serotype will transduce most effectively. For example, other AAV serotypes such as 4 and 5 are not dependent on heparan sulfate for entry into target cells, and therefore may provide a better vehicle for gene delivery to the cochlea. In addition, AAV-5 has recently been shown to use sialic acid as a coreceptor for entry into target cells. Hair cells are thought to express sialic acid on their surface, suggesting that AAV-5 may serve as an efficient gene delivery vehicle to these cells. Another critical component to these studies will be to compare transduction efficiencies between normal and gentamicin-damaged cochleae. We have chosen to use the mouse for the development of this transgene delivery system so that future gene transfer studies can take advantage of the numerous mouse mutations that have been shown to influence auditory development and function. These feasibility studies represent a new direction for our laboratory and will form the basis for the development of a full-scale research program. The focus of these future studies will be on delivering specific genes to the auditory system that potentially play a role in both the functional recovery of the cochlea, as well as neuroprotection following cochlear damage.